: Cells have multiple damage-response pathways that are activated by a radiation challenge. A p53-mediated pathway arrests cells in late G1, to temporarily prevent entry into S phase or to shunt them toward apoptosis. A second pathway prevents G cells from undergoing mitosis. However, neither of these cell cycle checkpoints can protect cells that are in the S phase at the time of radiation from replicating damaged templates. The P.I. has utilized a 2-D gel replicon mapping strategy to show that ionizing radiation specifically down-regulates initiation in the early firing dihydrofolate reductase (DHFR) origin in CHO cells and demonstrated that this S-phase damage-sensing (SDS) pathway also inhibits initiation in mid-firing rDNA origins in human cells, and therefore must function throughout the S period. The SDS pathway appears p53- (and probably pRB-) independent. The ori-b and or-g origins in the CHO DHFR domain have been characterized, and each contains a pronounced micrococcal nuclease hypersensitive site that presumably signals the presence of an origin-binding protein complex. This site is present in the pre-replicative (G1) state, but disappears after the origin fires in early S. It is proposed that modification of this protein/DNA interaction is the ultimate step in the SDS pathway that specifically inhibits initiation in response to radiation. This could occur by direct modification of an origin-binding protein (or a protein that interacts with it), or by a global effect on chromatin structure that indirectly affects the activity of this complex. In this application, a comprehensive analysis of the chromatin in the ori-beta and ori-gamma loci, as well as in the DHFR replicon as a whole is proposed. These experiments are proposed to distinguish between the local versus global possibilities. Should specific origin-binding protein modification be suggested, a one hybrid screening method for identifying the cDNA encoding the specific protein will be performed. Should more global modifications be indicated, experiments to determine whether these alterations are effected by altering association of the replicon with the nuclear matrix are described. The long-range goals are to understand the SDS pathway at the molecular level, starting at a defined origin and its effectors and working backward in the pathway. Since loss of this pathway in Ataxia Telangiectasia renders cells exquisitely sensitive to ionizing radiation, we hope to develop novel radiation sensitization strategies by manipulation of the pathway.